Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosus
Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosus
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DOI：
10.1107/s1744309105028393
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发表时间：
2005-10-01
影响因子：
0.9
通讯作者：
Hagen, WR
中科院分区：
文献类型：
--
作者：
Agapay, RC;Savvides, SN;Hagen, WR
RAM 结构域是原核生物中存在的几种配体结合模块之一，推测其调节特定基因的转录。迄今为止，尚未报道此类模块的水解活性。奇怪的是，在针对显色酯的水解活性筛选过程中，分离出了强烈火球菌中的一个独立 RAM 结构域。编码该蛋白质的基因在大肠杆菌中被克隆和表达，并在单一纯化步骤后结晶。使用传统 X 射线源获得分辨率为 2.8 埃的晶体 X 射线衍射数据。使用同步辐射，重组蛋白与 1,2-环氧-3-(4-硝基苯氧基)丙烷 (EPNP) 和苯甲基磺酰氟 (PMSF) 共结晶产生的晶体分别产生 2.2 和 2.8 埃的数据。未处理和EPNP处理的晶体均在C2空间群中同晶结晶，并且在不对称单元中包含三个二聚体。经过 PMSF 处理的晶体也属于该空间群，并且具有几乎相同的堆积密度，但表现出显着不同的晶胞参数。
The RAM domain is one of several ligand-binding modules present in prokaryotes that are presumed to regulate the transcription of specific genes. To date, no hydrolytic activity has been reported for such modules. Curiously, a stand-alone RAM domain in Pyrococcus furiosus was isolated during a screen for hydrolytic activity against chromogenic esters. The gene encoding this protein was cloned and expressed in Escherichia coli and crystallized after a single purification step. X-ray diffraction data from the crystals were obtained to a resolution of 2.8 angstrom using a conventional X-ray source. The cocrystallization of the recombinant protein with 1,2-epoxy-3-(4-nitrophenoxy) propane (EPNP) and phenylmethylsulfonyl fluoride (PMSF) produced crystals that yielded data to 2.2 and 2.8 angstrom, respectively, using synchrotron radiation. Both the untreated and EPNP-treated crystals crystallize isomorphously in space group C2 and contain three dimers in the asymmetric unit. The PMSF-treated crystals also belong to this space group and have almost identical packing density, but show dramatically different unit-cell parameters.